The present invention is related to the bioconversion of a carbon source to vanillin and more particularly, to methods of producing vanillin from a carbon source by microbe-catalyzed conversion of the carbon source to vanillic acid and enzyme-catalyzed reduction of vanillic acid to produce vanillin.
Natural vanillin is produced from glucovanillin (FIG. 1) when the beans of the orchid Vanilla planifolia are submitted to a multi-step curing process. Ranadive, A. S., In Spices, Herbs, and Edible Fungi, Charalambous, G., Ed., Elsevier: Amsterdam, p. 517 (1994). Because of the extreme care that must be exercised during vine cultivation, bean harvesting, and hand pollination of flowers, natural vanillin can supply only 2xc3x97104 kg/yr of the world""s 1.2xc3x97107 kg/yr demand for vanillin. Clark, G. S., Perfum. Flavor. 15:45 (1990). This has resulted in substitution of synthetic vanillin for natural vanilla in most flavoring applications. Condensation of glyoxylic acid with benzene-derived guaiacol (FIG. 1) is therefore currently the dominant route for vanillin manufacture. Ranadive, A. S., In Spices, Herbs, and Edible Fungi, Charalambous, G., Ed., Elsevier: Amsterdam, p. 517 (1994); Clark, G. S., Perfum. Flavor. 15:45 (1990); Esposito, L. et al., Kirk-Othmer Encyclopedia of Chemical Technology, Fourth Ed., Kroschwitz, J. I.; Howe-Grant, M., Ed.; Wiley: New York, Vol. 24:812 (1997). Limited vanilla bean supplies have also led to extensive research into the use of plant tissue culture and microbes to convert ferulic acid (FIG. 1) into vanillin suitable for labelling as a natural or nature-equivalent flavoring. Falconnier, B. et al., J. Biotechnol. 37:123 (1994); Lesage-Meessen, L. et al., J. Biotechnol. 50:107 (1996); Lesage-Meessen, L. et al., Appl. Microbiol. Biotechnol. 47:393 (1997); Labuda, I. M. et al., U.S. Pat. No. 5,279,950 (1994); Westcott, R. J. et al., Phytochemistry 35:135 (1994).
Vanillin is second only to aspartame in terms of market size for a food additive. Vanilla extract derived from V. planifolia pods has the advantage of being labeled as a natural flavoring. However, as described above, only relative small volumes of vanilla flavoring can be derived from V. planifolia cultivation. Synthesis of vanillin from benzene-derived guaiacol is therefore the basis of large-scale industrial manufacture of vanillin. This vanillin however, can not be labeled as a natural flavoring and synthesis of vanillin from benzene-derived guaiacol is not environmentally benign. With respect to the ferulate-derived vanillin, although it can be labeled as a natural flavoring, the microbes and cultured plant cells used to process the ferulic acid give low titers of vanillin (approximately 1 g/L). Falconnier, B. et al., J. Biotechnol. 37:123 (1994); Lesage-Meessen, L. et al., J. Biotechnol. 50:107 (1996); Lesage-Meessen, L. et al., Appl. Microbiol. Biotechnol. 47:393 (1997); Labuda, I. M. et al., U.S. Pat. No. 5,279,950 (1994); Westcott, R. J. et al., Phytochemistry35:135 (1994). A further problem is the availability of ferulic acid; although corn fiber is rich in ferulic acid esters, no process amenable to commercial scale isolation and processing of this ferulic acid has been developed.
It would thus be desirable to provide a method for synthesizing vanillin. It would further be desirable to provide a method for synthesizing vanillin which is economically attractive. It would also be desirable to provide a method for synthesizing vanillin which is environmentally benign. It would further be desirable to provide a method for synthesizing vanillin which utilizes an abundant, renewable resource as the starting material.
A bioengineered synthesis scheme for the production of vanillin from a carbon source is provided. In one embodiment, the bioconversion methods of the present invention comprise the steps of microbe-catalyzed conversion of a carbon source to vanillic acid followed by enzyme-catalyzed reduction of vanillic acid to produce vanillin. As shown in the synthesis scheme of FIG. 2, the microbe-catalyzed conversion step of the present invention requires five enzymes which are provided by a recombinant microbe. In a preferred embodiment, the recombinant microbe is Escherichia coli designed to cause dehydration of 3-dehydroshikimic acid and regioselective methylation of the resulting protocatechuic acid. The enzyme-catalyzed reduction step of the present invention comprises the reduction of vanillic acid to vanillin by aryl-aldehyde dehydrogenase. In a preferred embodiment, the aryl-aldehyde dehydrogenase is purified from Neurospora crassa. 
The biocatalytic synthesis of vanillin provided herein is environmentally benign, economically attractive, and utilizes abundant renewable sources, as starting materials.
Additional objects, advantages, and features of the present invention will become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings.